Methods and compositions for the extraction and amplification of nucleic acid from a sample

ABSTRACT

Provided herein are methods, compositions and kits to extract and relatively enrich by physical separation or amplification short base pair nucleic acid in the presence of a high background of genomic material (e.g., host or maternal nucleic acids).

RELATED PATENT APPLICATIONS

This patent application is a continuation of U.S. patent applicationSer. No. 14/180,810, filed on Feb. 14, 2014, entitled “Methods andCompositions for the Extraction and Amplification of Nucleic Acid from aSample,” naming Carolyn R. Hoyal-Wrightson, Andreas Braun, and KarstenE. Schmidt as inventors; which is a divisional of U.S. patentapplication Ser. No. 12/301,985, now U.S. Pat. No. 8,679,741, filed onAug. 9, 2010, entitled “Methods and Compositions for the Extraction andAmplification of Nucleic Acid from a Sample,” naming Carolyn R.Hoyal-Wrightson, Andreas Braun, and Karsten E. Schmidt as inventors;which is a national stage of international patent application numberPCT/US2007/069991, filed on May 30, 2007, entitled “Methods andCompositions for the Extraction and Amplification of Nucleic Acid From aSample,” naming Carolyn R. Hoyal-Wrightson, Andreas Braun, and KarstenE. Schmidt as inventors; which claims the benefit of U.S. provisionalpatent application Nos. 60/810,228 and 60/807,061, filed on May 31, 2006and Jul. 11, 2006, respectively, each entitled “Methods and Compositionsfor the Extraction and Amplification of Nucleic Acid from a Sample,”respectively. The entire content of each of the foregoing patentapplications hereby is incorporated by reference herein, including alltext, drawings and tables, in jurisdictions providing for suchincorporation.

SEQUENCE LISTING

The instant patent application contains a Sequence Listing that has beensubmitted via EFS-Web and is hereby incorporated by reference in itsentirety. The ASCII copy, created on Jan. 23, 2009, is namedSEQ-6001-US.txt and is 1,234 bytes in size.

FIELD OF THE INVENTION

The invention relates to methods and kits for the extraction, andoptionally the amplification, of nucleic acids from a sample,particularly from a biological sample containing cell-free nucleicacids. The methods of the invention may be used in a wide range ofapplications, including the extraction of fetal nucleic acids frommaternal plasma, the detection of circulating nucleic acids fromneoplasms (malignant or non-malignant), the detection of early onset oftissue rejection, or any other application requiring the selectiveseparation of nucleic acids based on their size and/or apoptotic origin.

BACKGROUND

The isolation and subsequent amplification of nucleic acids play acentral role in molecular biology. Isolated, purified nucleic acids maybe used, inter alia, as a starting material for diagnosis and prognosisof diseases or disorders. Therefore, the isolation of nucleic acids,particularly by non-invasive means, is of particular importance for usein genetic analyses.

Current methods for the extraction of nucleic acids include the use oforganic-based methods (e.g., phenol/chloroform/isoamyl alcohol), orcapitalize upon ion interaction of nucleic acids in an aqueous solution(e.g., salting out in combination with alcohol, solution pH andtemperature) alone or in combination with anion exchange chromatographyor cation exchange chromatography. Organic-based methods employ the useof phenol/chloroform/isoamyl alcohol or variations thereof for isolatingDNA, but have serious disadvantages, namely the processes are verytime-consuming, require considerable experimental effort, and areassociated with an acute risk of exposure to toxic substances to thosecarrying out the isolation. Chromatography-based methods increaseflexibility and automation since these methods can be used incombination with multiple matrices (e.g., membranes, latex, magneticbeads, micro-titer plate, etc.) and in the presence or absence ofligands (e.g., DEAE, silica, acrylamide, etc.). However, these methodsare better suited to extract larger strands of nucleic acids to ensuregreater success in downstream analysis.

Previously, the recovery of smaller, fragmented nucleic acids frombiological samples was considered unimportant, and extraction methodswere designed to isolate large, undegraded nucleic acid molecules.Recently, however, it is shorter base pair nucleic acids (e.g., highlydegraded RNA or mRNA and apoptotic DNA) that have been shown to behighly informative for a wide range of applications, including prenataldiagnostics and the study of apoptotic DNA from host or non-hostsources. Methods to capture and protect RNA during extraction are nowcommon; however the ability to successfully analyze short, fragmentedDNA in the presence of more abundant, longer DNA has remained elusive.

SUMMARY OF THE INVENTION

There is a need for improved extraction methods capable of capturingsmall nucleic acid molecules. At the same time, these methods need to besimple, cost-effective and automatable in order to prove useful in theresearch and clinical environments. Thus, in one aspect, the inventionrelates to compositions, methods and kits for the extraction,amplification and analysis of nucleic acids based on their size. Studieshave shown that the majority of cell-free nucleic acid resulting fromneoplasms, allograft rejection, autoimmune reactions, fetal tissue, etc.has a relatively small size of approximately 1,200 base pairs or less,whereas the majority of cell-free nucleic acid arising in the host fromnon-programmed cell death-associated events has a size greater thanapproximately 1,200 base pairs.

The present invention, therefore, provides compositions, methods andkits for the enrichment, based on size discrimination, of nucleic acidof approximately 1,200 base pairs or less (herein referred to as “targetnucleic acid”) in a high background of genomic nucleic acid (hereinreferred to as “non-target nucleic acid”). This leads to a relativelyenriched fraction of nucleic acid that has a higher concentration ofsmaller nucleic acid.

The present invention provides methods for extracting target nucleicacid from a biological sample containing a mixture of non-target nucleicacid based on the size of the nucleic acid, wherein the target nucleicacid size is less than the size of the non-target nucleic acid in themixture, comprising the steps of introducing the biological sample to afirst extraction method designed to isolate non-target nucleic acid,wherein the target nucleic acid is not substantially isolated, therebycreating a supernatant that contains target nucleic acid; removing thesupernatant and introducing said supernatant to a second extractionmethod designed to isolate target nucleic acid, and, optionally, elutingthe target nucleic acid with an elution buffer suitable for elutingnucleic acid, whereby the target nucleic acid has been selectivelyextracted from the sample.

In another embodiment, the present invention provides compositions,methods and kits for the adsorption of target nucleic acid to a solidsupport in the presence of increasing concentrations of salt, wherebythe target nucleic acid is selectively enriched based on its molecularsize. The compositions and methods may be used to extract and enrich theamount of normally trace nucleic acid, which is initially in thepresence of high amounts of non-desired background nucleic acid, tolevels suitable for detection and analysis. The invention providescompositions and methods for binding nucleic acid under specificconditions to introduce size selection with the purpose of extraction ofany nucleic acid within the range of about 10 bases to about 5000 bases.

Nucleic acids are known to bind to a solid phase in the presence of achaotropic agent (see U.S. Pat. No. 5,234,809, which is herebyincorporated by reference). Thus, provided herein are improved methodsfor extracting low molecular weight nucleic acid in a sample by bringinga nucleic acid-containing solution to a low salt concentration state;adsorbing the nucleic acid to a solid support and separating the solidsupport from the solution; bringing the solution to a high saltconcentration state; adsorbing the nucleic acid to a solid support andseparating the solid support from the solution; and eluting adsorbednucleic acid from the solid support, whereby the low molecular weightnucleic acid has been selectively enriched from the sample.

In a related embodiment, the invention provides a method for extractingtarget nucleic acid from a biological sample containing a mixture ofnon-target nucleic acid based on the size of the nucleic acid, whereinthe target nucleic acid size is less than the size of the non-targetnucleic acid in the mixture, comprising the steps of mixing saidbiological sample, a salt and a nucleic acid binding solid support,wherein the salt is present at a concentration sufficient to bindnon-target nucleic acid, while binding substantially little to no targetnucleic acid, thereby creating a first binding solution; adsorbing thenon-target nucleic acid to the solid support, and separating the solidsupport from the solution; removing the supernatant of the first bindingsolution, and mixing said supernatant with additional salt and a nucleicacid binding solid support, wherein the salt is present at aconcentration sufficient to bind the target nucleic acid, therebycreating a second binding solution; adsorbing the target nucleic acid tothe solid support, and separating the solid support from the secondbinding solution, thereby creating a solid support-target nucleic acidcomplex; and eluting the adsorbed target nucleic acid from the solidsupport with an elution buffer suitable for eluting nucleic acid,whereby the target nucleic acid has been selectively extracted from thesample.

The methods of the present invention may be used to extract nucleic acidwithin the range of about 10 bases to about 5000 bases. In a preferredembodiment, the target nucleic acid is at least about 25 base pairs, butless than about 1200 base pairs, and can be between about 200 base pairsand about 600 base pairs.

The present invention relates to extracting nucleic acids such as DNA,RNA, mRNA, oligonucleosomal, mitochondrial, epigenetically modified,single-stranded, double-stranded, circular, plasmid, cosmid, yeastartificial chromosomes, artificial or man-made DNA, including unique DNAsequences, and DNA that has been reverse transcribed from an RNA sample,such as cDNA, and combinations thereof. In a preferred embodiment, thenucleic acid is cell-free nucleic acid. In another embodiment, thenucleic acids are derived from apoptotic cells. In another embodiment,the target nucleic acid is of fetal origin, and the non-target nucleicacid is of maternal origin.

The present invention relates to extracting nucleic acid from abiological sample such as whole blood, serum, plasma, umbilical cordblood, chorionic villi, amniotic fluid, cerbrospinal fluid, spinalfluid, lavage fluid (e.g., bronchoalveolar, gastric, peritoneal, ductal,ear, athroscopic) biopsy sample, urine, feces, sputum, saliva, nasalmucous, prostate fluid, semen, lymphatic fluid, bile, tears, sweat,breast milk, breast fluid, embryonic cells and fetal cells. In apreferred embodiment, the biological sample is plasma. In anotherpreferred embodiment, the biological sample is cell-free orsubstantially cell-free. In a related embodiment, the biological sampleis a sample of previously extracted nucleic acids.

The present invention is particularly useful for extracting fetalnucleic acid from maternal plasma. In a preferred embodiment, thebiological sample is from an animal, most preferably a human. In anotherpreferred embodiment, the biological sample is from a pregnant human. Ina related embodiment, the biological sample is collected from a pregnanthuman after the fifth week of gestation. In another embodiment, thepregnant human has a relatively elevated concentration of free fetalnucleic acid in her blood, plasma or amniotic fluid. In anotherembodiment, the pregnant human has a relatively decreased concentrationof apoptotic nucleic acid in her blood, plasma or amniotic fluid. Themethods of the present invention may be performed in conjunction withany known method to elevate fetal nucleic acid in maternal blood, plasmaor amniotic fluid. Likewise, the methods of the present invention may beperformed in conjunction with any known method to decrease apoptoticnucleic acid in maternal blood, plasma or amniotic fluid.

The present invention is based on the ability of nucleic acid toreversibly bind to a nucleic acid-binding solid support in the presenceof a salt, such as guanidine salt, sodium iodide, potassium iodide,sodium thiocyanate, urea, sodium chloride, magnesium chloride, calciumchloride, potassium chloride, lithium chloride, barium chloride, cesiumchloride, ammonium acetate, sodium acetate, ammonium perchlorate orsodium perchlorate, for example. In a preferred embodiment, the salt isa guanidine salt, most preferably guanidine (iso)thiocyanate, or is asodium salt, most preferably sodium perchlorate. In the methods providedherein, the salt is introduced at a concentration to bind nucleic acidto a solid support. In the first binding solution, a salt is added toyield a solution with a concentration in the range of 10 to 30% weightper volume capable of binding non-target nucleic acid, while minimizingthe binding of target nucleic acid. In a preferred embodiment, thenon-target nucleic acid is at least 1200 base pairs. In the secondbinding solution, a chaotropic substance is added to yield a solutionwith a salt concentration greater than 10%, and preferably in the rangeof 20 to 60% weight per volume, which is capable of binding targetnucleic acid.

In a related embodiment, the solid support is a hydroxyl donor (e.g.,silica or glass) or contains a functional group that serves as ahydroxyl donor and is attached to a solid support. Examples of solidsupports include paramagnetic microparticles, silica gel, silicaparticles, controlled pore glass, magnetic beads, biomagnetic separationbeads, microspheres, divinylbenzene (DVB) resin, cellulose beads,capillaries, filter membranes, columns, nitrocellulose paper, flatsupports, glass surfaces, metal surfaces, plastic materials, multiwellplates or membranes, wafers, combs, pins and needles, or any combinationthereof, for example. In a preferred embodiment, the solid support ismodified to reversibly bind nucleic acid. In another preferredembodiment, the solid support is a silica gel membrane.

In a related embodiment, the nucleic acid-solid support interaction isan electrostatic interaction. In another embodiment, the nucleicacid-solid support interaction is a polar interaction.

In a related embodiment, the solid support has a functional group-coatedsurface. In a preferred embodiment, the functional group-coated surfaceis silica-coated, hydroxyl coated, amine-coated, carboxyl-coated orencapsulated carboxyl group-coated, for example. A bead may besilica-coated or a membrane may contain silica gel in certainembodiments.

In the present invention, it is necessary to separate the nucleicacid-coated solid support from the first or second binding solutions.The solid support (e.g., silica-coated magnetic bead) can be separatedfrom the solutions by any method known in the art, including applying amagnetic field, applying vacuum filtration and/or centrifugation, or anycombination thereof. In a preferred embodiment, paramagnetic beads areseparated from one or both solutions using magnets or magnetic devices.

The methods provided herein may also be modified to introduce additionalsteps, for example, in order to improve the extraction of nucleic acidor improve analysis of target nucleic acid following extraction. Forexample, the biological sample may be first lysed in the presence of alysis buffer, which may comprise a chaotropic substance (e.g., salt), aproteinase, a protease or a detergent, or combinations thereof, forexample. The lysis step and the creation of the first binding solutionmay be performed simultaneously at a salt concentration sufficient tosolubilize or precipitate non-nucleic acid material (e.g., protein) inthe sample and to bind the non-target nucleic acid to the solid support.In another embodiment, the method includes adding a washing step orsteps to remove non-nucleic acid from the solid-support-target nucleicacid complex. In another embodiment, the solid support-target nucleicacid complex is further washed successively with a wash buffer and oneor more alcohol-water solutions, and subsequently dried. In a preferredembodiment, the wash buffer comprises a chaotropic substance (e.g.,salt), and optionally, a carrier such as LPA, RNA, tRNA, dextran blue,glycogen or polyA RNA, for example. In another embodiment, the secondbinding solution also comprises a carrier such as LPA, RNA, tRNA,dextran blue, glycogen or polyA RNA, for example.

The methods provided herein may also be modified to combine steps, forexample, in order to improve automation. For example, mixing the firstbinding solution and adsorbing the non-target nucleic acid to the solidsupport may be performed simultaneously. Likewise, mixing the secondbinding solution and adsorbing the target nucleic acid to the solidsupport may be performed simultaneously.

In another embodiment, the methods provided herein may be performedprior to, subsequent to, or simultaneously with another method forextracting nucleic acid such as electrophoresis, liquid chromatography,size exclusion, microdialysis, electrodialysis, centrifugal membraneexclusion, organic or inorganic extraction, affinity chromatography,PCR, genome-wide PCR, sequence-specific PCR, methylation-specific PCR,introducing a silica membrane or molecular sieve, and fragment selectiveamplification.

The present invention also further relates to a kit comprising reagentsfor a first binding buffer formulated to comprise a suitable salt,wherein the salt is present at a concentration appropriate for binding anon-target nucleic acid characterized by a particular size, to the solidsupport; a second binding buffer formulated to comprise a suitable salt,wherein the salt is present at a concentration appropriate for binding atarget nucleic acid characterized by a particular size, to the solidsupport; an aqueous solution of functional group-coated paramagneticmicroparticles; and instructions for performing the target nucleic acidextraction. In another embodiment, the kit additionally comprisesreagents for the formulation of a wash buffer and an elution buffer,wherein the wash buffer dissolves impurities, but not nucleic acidsbound to solid support and the elution buffer is a non-salt bufferedsolution with a pH range between about 7.0 to 8.5.

The present invention also provides methods for a post purificationprocess which allows enrichment of target nucleic acid by ligation-basedmethods followed by amplification. In one embodiment of the invention,the present invention provides a method for selectively amplifying atarget nucleic acid from a biological sample containing a mixture ofnon-target nucleic acid, wherein the target nucleic acid is a doublestranded, blunt end nucleic acid fragment with 5′ phosphorylated ends,comprising the steps of a) mixing the biological sample, a 5′ adapterand a 3′ adapter, wherein the 3′ adapter is complementary to the 5′adapter at the 3′ end and thus capable of creating a double-strandedadapter complex; b) introducing a ligase to the mixture of step a) andligating the 5′ adapter of the double-stranded adapter complex to thetarget nucleic acid, thereby creating a ligated sample; c) heatingligated sample to release the 3′ adapter; d) adding a polymerase to fillin the single-stranded 5′ protruding ends; and e) adding 5′ adapterprimers to amplify the target nucleic acid. In a related embodiment, themethod includes the additional step of performing target-specificamplification using target-specific primers. In another embodiment, adideoxy-nucleotide is incorporated into the 3′ position of the 3′adapter. In another embodiment, the 5′ adapters of step a) are bound toa solid support. Optionally, spacer arms are introduced between the 5′adapter at the 5′ end and the solid support. In another embodiment,solid support-bound ligation products are combined with the non-solidsupport products of claim 1 prior to amplification step e).

In another embodiment of the invention, a method is provided thatselectively detects and amplifies target nucleic acid using acombination of the following 3 steps: 1) treating total isolated nucleicacid from a biological sample with a ligase that can covalently joinblunt 5′-phosphorylated nucleic acid ends (e.g. T4 or T7 DNA ligase)under conditions that favor unimolecular circularization of the nucleicacid molecules; 2) amplifying the nucleic acid with target-specificprimers and a method that is selective for circular nucleic acid, forexample, either a) via a rolling circle amplification withtarget-specific primers, or b) via inverse PCR with target-specificprimers for the gene of interest; and 3) characterizing the amplifiednucleic acid by direct or indirect qualitative and/or quantitativemolecular characterization methods.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the successful extraction of low base pair DNA from a 1 kbDNA ladder (Promega™) in the presence of guanidine thiocyanate (GuSCN).

FIG. 2 shows the successful extraction of low base pair DNA from a 1 kbDNA ladder (Promega™) in the presence of sodium perchlorate (NaClO₄).

FIG. 3 is a schematic showing the steps of adapter mediated ligation forthe selective detection and amplification of target nucleic acids.

FIG. 4 is a schematic showing the steps of circular ligation and inversePCR for the selective detection and amplification of target nucleicacids.

FIG. 5 is a schematic showing the steps of rolling circle amplification(RCA) for the selective detection and amplification of target nucleicacids.

DETAILED DESCRIPTION OF THE INVENTION

The presence of cell-free nucleic acid in peripheral blood is a wellestablished phenomenon. While cell-free nucleic acid may originate fromseveral sources, it has been demonstrated that one source of circulatingextracellular nucleic acid originates from programmed cell death, alsoknown as apoptosis. The source of nucleic acid that arise as a result ofapoptosis may be found in many body fluids and originate from severalsources, including, but not limited to, normal programmed cell death inthe host, induced programmed cell death in the case of an autoimmunedisease, septic shock, neoplasms (malignant or non-malignant), ornon-host sources such as an allograft (transplanted tissue), or thefetus or placenta of a pregnant woman. The applications for thedetection, extraction and relative enrichment of extracellular nucleicacid from peripheral blood or other body fluids are widespread and mayinclude inter alia, non-invasive prenatal diagnosis, cancer diagnostics,pathogen detection, auto-immune response and allograft rejection.

The present invention includes methods, compositions and kits to extractand relatively enrich by physical separation or amplification short basepair nucleic acid in the presence of a high background of genomicmaterial (e.g., host or maternal nucleic acids). More specifically, thepresent invention provides compositions, methods and kits for theselective extraction and relative enrichment, based on sizediscrimination, of nucleic acid of approximately 1,200 base pairs orless (herein referred to as “target nucleic acid”) in a high backgroundof genomic nucleic acids (herein referred to as “non-target nucleicacid”). This leads to a relatively enriched fraction of nucleic acidthat has a higher concentration of smaller nucleic acids.

The methods of the present invention may be used to improve pathogendetection. Methods for rapid identification of unknown bioagents using acombination of nucleic acid amplification and determination of basecomposition of informative amplicons by molecular mass analysis aredisclosed and claimed in published U.S. Patent applications 20030027135,20030082539, 20030124556, 20030175696, 20030175695, 20030175697, and20030190605 and U.S. patent application Ser. Nos. 10/326,047,10/660,997, 10/660,122 and 10/660,996, all of which are incorporatedherein by reference in entirety.

The term “host cell” as used herein is any cell into which exogenousnucleic acid can be introduced, producing a host cell which containsexogenous nucleic acid, in addition to host cell nucleic acid. As usedherein the terms “host cell nucleic acid” and “endogenous nucleic acid”refer to nucleic acid species (e.g., genomic or chromosomal nucleicacid) that are present in a host cell as the cell is obtained. As usedherein, the term “exogenous” refers to nucleic acid other than host cellnucleic acid; exogenous nucleic acid can be present into a host cell asa result of being introduced in the host cell or being introduced intoan ancestor of the host cell. Thus, for example, a nucleic acid specieswhich is exogenous to a particular host cell is a nucleic acid specieswhich is non-endogenous (not present in the host cell as it was obtainedor an ancestor of the host cell). Appropriate host cells include, butare not limited to, bacterial cells, yeast cells, plant cells andmammalian cells.

The term “extraction” as used herein refers to the partial or completeseparation and isolation of a nucleic acid from a biological ornon-biological sample comprising other nucleic acids. The terms“selective” and “selectively” as used herein refer to the ability toextract a particular species of nucleic acid molecule, on the basis ofmolecular size from a combination which includes or is a mixture ofspecies of nucleic acid molecules.

The terms “nucleic acid” and “nucleic acid molecule” may be usedinterchangeably throughout the disclosure. The terms refer to adeoxyribonucleotide (DNA), ribonucleotide polymer (RNA), RNA/DNA hybridsand polyamide nucleic acids (PNAs) in either single- or double-strandedform, and unless otherwise limited, would encompass known analogs ofnatural nucleotides that can function in a similar manner as naturallyoccurring nucleotides.

The term “target nucleic acid” as used herein refers to the nucleic acidof interest that is extracted based on its molecular size, preferably ina second extraction step, and further isolated for downstream analysis.In a preferred embodiment, the target nucleic acid has a molecular sizesmaller than the non-target nucleic acid present in the biologicalsample, for example, smaller than 1200 base pairs. In a relatedembodiment, the target nucleic acid is from apoptotic DNA, fetal DNA,oncogenic DNA, or any non-host DNA. In another related embodiment, thetarget nucleic acid is cell-free nucleic acid. In another relatedembodiment, the target nucleic acid is oligonucleosomal nucleic acidgenerated during programmed cell death.

The term “non-target nucleic acid” as used herein refers to therelatively high amount of non-desired background nucleic acid present ina biological sample, which is extracted, preferably, in a firstextraction step. In a preferred embodiment, non-target nucleic acid hasa molecular size larger than target nucleic acid, for example, greaterthan 1200 base pairs. In a related embodiment, non-target nucleic acidis from a host or host cell. In a preferred embodiment, non-targetnucleic acid is of maternal origin.

The term “molecular size” as used herein refers to the size of a nucleicacid molecule, which may be measured in terms of a nucleic acidmolecule's mass or length (bases or base pairs).

Fetal nucleic acid is present in maternal plasma from the firsttrimester onwards, with concentrations that increase with progressinggestational age (Lo et al. Am J Hum Genet (1998) 62:768-775). Afterdelivery, fetal nucleic acid is cleared very rapidly from the maternalplasma (Lo et al. Am J Hum Genet (1999) 64:218-224). Fetal nucleic acidis present in maternal plasma in a much higher fractional concentrationthan fetal nucleic acid in the cellular fraction of maternal blood (Loet al. Am J Hum Genet (1998) 62:768-775). Thus, in another embodiment,the target nucleic acid is of fetal origin, and the non-target nucleicacid is of maternal origin.

The present invention relates to extracting nucleic acid from abiological sample such as whole blood, serum, plasma, umbilical cordblood, chorionic villi, amniotic fluid, cerbrospinal fluid, spinalfluid, lavage fluid (e.g., bronchoalveolar, gastric, peritoneal, ductal,ear, athroscopic), biopsy sample, urine, feces, sputum, saliva, nasalmucous, prostate fluid, semen, lymphatic fluid, bile, tears, sweat,breast milk, breast fluid, embryonic cells and fetal cells. In apreferred embodiment, the biological sample is blood, and morepreferably plasma. As used herein, the term “blood” encompasses wholeblood or any fractions of blood, such as serum and plasma asconventionally defined. Blood plasma refers to the fraction of wholeblood resulting from centrifugation of blood treated withanticoagulants. Blood serum refers to the watery portion of fluidremaining after a blood sample has coagulated. In a preferred method,blood handling protocols are followed to ensure minimal degradation ofnucleic acid in the sample and to minimize the creation of apoptoticnucleic acid in the sample. Blood handling methods are well known in theart.

In another preferred embodiment, the biological sample is cell-free orsubstantially cell-free. In a related embodiment, the biological sampleis a sample containing previously extracted, isolated or purifiednucleic acids. One way of targeting target nucleic acid is to use thenon-cellular fraction of a biological sample; thus limiting the amountof intact cellular material (e.g., large strand genomic DNA) fromcontaminating the sample. In an embodiment of the invention, a cell-freesample such as pre-cleared plasma, urine, etc. is first treated toinactivate intracellular nucleases through the addition of an enzyme, achaotropic substance, a detergent or any combination thereof. In anotherembodiment, the biological sample is first treated to removesubstantially all cells from the sample by any of the methods known inthe art, for example, centrifugation, filtration, affinitychromatography, etc.

The term “concentration sufficient to selectively bind” as used hereinrefers to an amount sufficient to cause at least 50%, more preferably70%, even more preferably 90% or more of the target nucleic acid to bindto an adsorptive surface. Suitable solid phase carriers include, but arenot limited to, other particles, fibers, beads and or supports whichhave an affinity for nucleic acids or may be modified (e.g., theaddition of a functional group or groups) to bind nucleic acids, andwhich can embody a variety of shapes, that are either regular orirregular in form, provided that the shape maximizes the surface area ofthe solid phase, and embodies a carrier which is amenable to microscalemanipulations. In a preferred embodiment, silica-coated magnetic beadsare used. In a preferred embodiment, the solid support is modified toreversibly bind nucleic acid. In a related embodiment, the solid supporthas a functional group-coated surface. In a preferred embodiment, thefunctional group-coated surface is silica-coated, hydroxyl-coated,amine-coated, carboxyl-coated and encapsulated carboxyl group-coated.

The term “functional group-coated surface” as used herein refers to asurface which is coated with moieties which reversibly bind nucleicacids. One example is a surface which is coated with moieties which eachhave a free functional group which is bound to the amino group of theamino silane or the solid support; as a result, the surfaces of thesolid support are coated with the functional group containing moieties.In one embodiment, the functional group is a carboxylic acid. A suitablemoiety with a free carboxylic acid functional group is a succinic acidmoiety in which one of the carboxylic acid groups is bonded to the amineof amino silanes through an amide bond and the second carboxylic acid isunbonded, resulting in a free carboxylic acid group attached or tetheredto the surface of the paramagnetic microparticle. Suitable solid phasecarriers having a functional group coated surface that reversibly bindsnucleic acid molecules are for example, magnetically responsive solidphase carriers having a functional group-coated surface, such as, butnot limited to, silica-coated, hydroxyl-coated, amino-coated,carboxyl-coated and encapsulated carboxyl group-coated magnetic beads.In another example, an oligonucleotide of the invention (e.g., anadapter or primer) is labeled with biotin which may bind to immobilizedstreptavidin.

The extraction of nucleic acid from biological material requires celllysis, inactivation of cellular nucleases and separation of the desirednucleic acid from cellular debris. Common lysis procedures includemechanical disruption (e.g., grinding, hypotonic lysis), chemicaltreatment (e.g., detergent lysis, chaotropic agents, thiol reduction),and enzymatic digestion (e.g., proteinase K). In the present invention,the biological sample may be first lysed in the presence of a lysisbuffer, chaotropic agent (e.g., salt) and proteinase or protease. Cellmembrane disruption and inactivation of intracellular nucleases may becombined. For instance, a single solution may contain detergents tosolubilise cell membranes and strong chaotropic salts to inactivateintracellular enzymes. After cell lysis and nuclease inactivation,cellular debris may easily be removed by filtration or precipitation.

In another embodiment, lysis may be blocked. In these embodiments, thesample may be mixed with an agent that inhibits cell lysis to inhibitthe lysis of cells, if cells are present, where the agent is a membranestabilizer, a cross-linker, or a cell lysis inhibitor. In some of theseembodiments, the agent is a cell lysis inhibitor, and may beglutaraldehyde, derivatives of glutaraldehyde, formaldehyde, formalin,or derivatives of formaldehyde. See U.S. patent application 20040137470,which is hereby incorporated by reference.

In another embodiment, the method includes adding a washing step orsteps to remove non-nucleic acid molecules, for example salts, from thesolid-support-target nucleic acid complex or surrounding solution.Non-nucleic acid molecules are then removed with an alcohol-based washand the target nucleic acid is eluted under low- or no-salt conditions(TE buffer or water) in small volumes, ready for immediate use withoutfurther concentration. In another embodiment, extraction is improved bythe introduction of a carrier such as tRNA, glycogen, polyA RNA, dextranblue, linear poly acrylamide (LPA), or any material that increases therecovery of nucleic acid. The carriers may be added to the secondbinding solution or washing buffer.

In another embodiment of the invention, the final relative percentage oftarget nucleic acid to non-target nucleic acid is at least about 5-6%fetal DNA, about 7-8% fetal DNA, about 9-10% fetal DNA, about 11-12%fetal DNA, about 13-14% fetal DNA. about 15-16% fetal DNA, about 16-17%fetal DNA, about 17-18% fetal DNA, about 18-19% fetal DNA, about 19-20%fetal DNA, about 20-21% fetal DNA, about 21-22% fetal DNA, about 22-23%fetal DNA, about 23-24% fetal DNA, about 24-25% fetal DNA, about 25-35%fetal DNA, about 35-45% fetal DNA, about 45-55% fetal DNA, about 55-65%fetal DNA, about 65-75% fetal DNA, about 75-85% fetal DNA, about 85-90%fetal DNA, about 90-91% fetal DNA, about 91-92% fetal DNA, about 92-93%fetal DNA, about 93-94% fetal DNA, about 94-95% fetal DNA, about 95-96%fetal DNA, about 96-97% fetal DNA, about 97-98% fetal DNA, about 98-99%fetal DNA, or about 99-99.7% fetal DNA.

The methods provided herein may also be modified to combine steps, forexample, in order to improve automation.

In another example, the methods of the present invention may be used inconjunction with any known technique suitable for the extraction,isolation or purification of nucleic acids, including, but not limitedto, cesium chloride gradients, gradients, sucrose gradients, glucosegradients, centrifugation protocols, boiling, Microcon 100 filter,Chemagen viral DNA/RNA 1k kit, Chemagen blood kit, Qiagen purificationsystems, Qiagen MinElute kits, QIA DNA blood purification kit, HiSpeedPlasmid Maxi Kit, QIAfilter plasmid kit, Promega DNA purificationsystems, MangeSil Paramagnetic Particle based systems, Wizard SVtechnology, Wizard Genomic DNA purification kit, Amersham purificationsystems, GFX Genomic Blood DNA purification kit, Invitrogen LifeTechnologies Purification Systems, CONCERT purification system, Mo BioLaboratories purification systems, UltraClean BloodSpin Kits, andUlraClean Blood DNA Kit.

In an embodiment of the invention, the first extraction method is anyknown or modified technique suitable for the extraction, isolation orpurification of non-target nucleic acids (i.e., larger than targetnucleic acids), including, but not limited to, cesium chloridegradients, gradients, sucrose gradients, glucose gradients,centrifugation protocols, boiling, Microcon 100 filter, Chemagen viralDNA/RNA 1k kit, Chemagen blood kit, Qiagen purification systems, QiagenMinElute kits, QIA DNA blood purification kit, HiSpeed Plasmid Maxi Kit,QIAfilter plasmid kit, Promega DNA purification systems, MangeSilParamagnetic Particle based systems, Wizard SV technology, WizardGenomic DNA purification kit, Amersham purification systems, GFX GenomicBlood DNA purification kit, Invitrogen Life Technologies PurificationSystems, CONCERT purification system, Mo Bio Laboratories purificationsystems, UltraClean BloodSpin Kits, and UlraClean Blood DNA Kit. In arelated embodiment, one or more of the above methods is modified toselectively extract larger, non-target nucleic acids while notextracting smaller, target nucleic acids. For example, the temperature,pH or reagent concentrations of one or more of the above methods may bemodified.

In another embodiment, the second extraction method is any known ormodified technique suitable for the extraction, isolation orpurification of target nucleic acids (i.e., smaller than non-targetnucleic acids), including, but not limited to, cesium chloridegradients, gradients, sucrose gradients, glucose gradients,centrifugation protocols, boiling, Microcon 100 filter, Chemagen viralDNA/RNA 1k kit, Chemagen blood kit, Qiagen purification systems, QiagenMinElute kits, QIA DNA blood purification kit, HiSpeed Plasmid Maxi Kit,QIAfilter plasmid kit, Promega DNA purification systems, MangeSilParamagnetic Particle based systems, Wizard SV technology, WizardGenomic DNA purification kit, Amersham purification systems, GFX GenomicBlood DNA purification kit, Invitrogen Life Technologies PurificationSystems, CONCERT purification system, Mo Bio Laboratories purificationsystems, UltraClean BloodSpin Kits, and UlraClean Blood DNA Kit. In arelated embodiment, one or more of the above methods is modified toselectively extract smaller nucleic acids, for example, present in asupernatant from a previously extracted sample. For example, thetemperature, pH or reagent concentrations of one or more of the abovemethods may be modified.

The present invention also further relates to kits for practicing themethods of the invention.

Ligation-Based Methods for Selective Nucleic Acid Detection andAmplification

Programmed cell death or apoptosis is an essential mechanism inmorphogenesis, development, differentiation, and homeostasis in allmulticellular organisms. Typically, apoptosis is distinguished fromnecrosis by activation of specific pathways that result incharacteristic morphological features including DNA fragmentation,chromatin condensation, cytoplasmic and nuclear breakdown, and theformation of apoptotic bodies.

Caspase-activated DNase (CAD), alternatively called DNA fragmentationfactor (DFF or DFF40), has been shown to generate double-stranded DNAbreaks in the internucleosomal linker regions of chromatin leading tonucleosomal ladders consisting of DNA oligomers of approximately 180base pairs or multiples thereof. The majority of the ladder fragments(up to 70%) occur as nucleosomal monomers of 180 bp. All fragments carry5′-phosphorylated ends and the majority of them are blunt-ended (Widlaket al, J Biol Chem. 2000 Mar. 17; 275(11):8226-32, which is herebyincorporated by reference). Since non-apoptotic DNA is lacking thisfeature, any method that can select for DNA fragments with blunt,5′-phosphorylated ends, is suitable to select for specific features(such as size, sequence and DNA base methylation differences) of theapoptotic DNA in a given biological sample. See for example, US patentapplications 20050019769, 20050164241, 20030044388, or 20060019278, allof which are hereby incorporated by reference.

Very short, single base 3′ and 5′-overhangs have also been detected butrepresent a minority of the DNA species in apoptotic ladders (Didenko etal, Am J Pathol. 2003 May; 162(5):1571-8; Widlak et al, 2000, both ofwhich are hereby incorporated by reference). Hence, methods that areselective for both, blunt, and 5′-phosphorylated blunt ends, are onlyslightly less sensitive but retain very high specificity for DNA ofapoptotic origin.

For enrichment and detection of apoptotic DNA ladders in mammaliantissues, a method has been described that takes advantage of thepresence of blunt, 5′-phosphorylated ends in apoptotic DNA by ligationof synthetic, blunt-ended linkers to both ends of linear apoptotic DNAfragments with T4 ligase which is able to form a covalent bond betweenthe 3′-hydroxy ends of the synthetic linker and the 5′-phosphorylatedends of the DNA fragments (Staley et al, Cell Death Differ. 1997January; 4(1):66-75, which is hereby incorporated by reference). Themethod can only be used as a generic tool to characterize the sizedistribution of apoptotic ladders in specific tissues in general, and isnot site or sequence specific.

A variation of the method, that employs biotinylated hairpin probesstained with fluorescence dye streptavidin conjugates had beenintroduced described patent (Didenko et al 1999; U.S. Pat. Nos.6,013,438 and 6,596,480, which are hereby incorporated by reference) toselectively detect terminal apoptotic activities in tissue sections.

Recently, the concept of blunt-end ligation-mediated whole genomeamplification of apoptotic and necrotic plasma DNA has been introduced(Li et al, J Mol Diagn. 2006 February; 8(1):22-30, which is herebyincorporated by reference) for the analysis of allelic imbalance intumor-specific DNA biomarkers. In this approach, isolated plasma DNA isfirst treated with T4 DNA polymerase to convert DNA fragments toblunt-ends before the blunt, 5′-phosphorylated DNA termini areself-ligated or cross ligated. The self-ligated, circular fragments arethen amplified approximately 1,000 fold via random primer-initiatedmultiple displacement amplification. However, since this approachamplifies all apoptotic DNA sequences present in the sample, at least 1ng (which represents about 300 genome equivalents of human DNA) isrequired to maintain equal genomic representation and gene-dosage andallelic ratios present before amplification.

Thus, there is an increasing need to characterize known mutations andepimutations of specific DNA fragments from specific cells or tissues orpresent as extracellular fragments in biological fluids in atarget-specific manner in the presence of high background of wild-typeDNA (e.g. somatic mutations of DNA from cells responding to a xenobioticof drug treatment; from inflamed, malignant or otherwise diseasedtissues; from transplants or from differences of fetal and maternal DNAduring pregnancy).

The present invention, therefore, provides a method for selectivelyamplifying short, fragmented nucleic acid by adapter mediated ligationand other related methods. The method capitalizes on the blunt end and5′-phosphorylated nature of the target nucleic acid as a means to attacha non-genome specific adapter to the blunt ends using a ligationprocess. While the nature of the termini of all cell-free nucleic acidis unknown, coupling this method with short extension times duringamplification will favor the amplification of the oligonucleosomemonomer and short multimers. Since the target nucleic acid is shorterthan the non-targeted nucleic acid, the target nucleic acid can beenriched over the non-target nucleic acid. This method can be furthercoupled with specific amplification of a nucleic acid region of interestfor further analysis. In the present invention, the 3′ and 5′dephosphorylated adapters are complementary and form a double-strandedblunt end adapter complex. The 5′ adapter of the adapter complex ligatesto the 5′ phosphorylated strand of the target nucleic acid, and heat isintroduced to release the shorter, unligated 3′ adapter. Next, the 5′protruding ends of the ligated complex are filled in by a thermostableDNA polymerase. The 5′ adapter is reintroduced and serves as a PCRprimer for whole genome amplification.

The method is particularly useful for detecting oligonucleosomes.Oligonucleosomes are the repeating structural units of chromatin, eachconsisting of approximately 200 base pairs of DNA wound around a histonecore that partially protects the DNA from nuclease digestion in vitroand in vivo. These units can be found as monomers or multimers andproduce what is commonly referred to as an apoptotic DNA ladder. Theunits are formed by nuclease digestion of the flanking DNA not bound tohistone resulting in the majority of oligonucleosomes being blunt endedand 5′-phosphorylated. In biological systems in which only a smallpercentage of cells are apoptotic, or in which apoptosis is occurringasynchronously, oligonucleosomes are hard to detect and harder toisolate; however, they can serve as predictors for disease and otherconditions (see US patent application 20040009518, which is herebyincorporated by reference).

The term “5′ dephosphorylated adapter” as used herein refers to anucleic acid which comprises about 20 to 30 base pairs that iscomplementary to a short dephosphorylated adapter and capable ofhybridizing thereto to form a double-stranded, blunt end adapter complexcapable of ligating to target nucleic acid. Specifically, the 5′adapterligates to the 5′phosphorylated base of the target nucleic acid.

The term “3′ dephosphorylated adapter” refers to a nucleic acid whichcomprises about 10 to 15 base pairs that is complementary to the 5′adapter at the 3′ end, thus capable of creating a double-strandedblunted end necessary for ligation. The 3′ adapter does not bind orligate to the oligonucleosomal DNA.

The term “5′ adapter primer” as used herein refers to the sameoligonucleotide sequence as the 5′ dephosphorylated adapter, but islater reintroduced to the ligated sample to facilitate the whole genomeamplification.

The term “adapter complex” as used herein refers to the hybridized,double-stranded 5′ adapter and 3′ adapter molecule.

The method is semi quantitative. By comparing the numbers of PCR cyclesneeded to detect target nucleic acid in two samples, the relative amountof target nucleic acid occurring in each sample can be estimated.

In another embodiment of the invention, the 5′ adapters are bound to asolid support for increased enrichment of the target nucleic acid. Inthis embodiment, non-ligated, non-target nucleic acid is substantiallyremoved from the solution, and amplification can proceed using only thetargeted material that has ligated to the 5′ adapters. This embodimentof the invention improves the enrichment of the target nucleic acid byremoving genomic non-target nucleic acid that may compete with thetarget nucleic acid in the target-specific amplification step. Forexample, in a maternal sample, if the target sequence is present in boththe mother and the fetus, and the maternal sample is very abundant(>95%), under normal circumstances the fetal nucleic acid would not bedetectable as it would be out-competed by the maternal nucleic acid inthe first cycles of amplification. If the fetal nucleic acid is part orwholly oligonucleosomal in nature, and the majority of the ligatedsample is fetal in nature, maternal nucleic acid is still present in thesample, which can compete with the fetal nucleic acid in thetarget-specific amplification step. Separation of non-target nucleicacid from the target nucleic acid (i.e., ligated sample) increases thedetection of fetal nucleic acid in cases where there is an abundance ofmaternal nucleic acid in the initial biological sample, there is sampledegradation, or there is a maternal condition (e.g., autoimmune disease,transplant rejection, cancer) that increases the amount of maternaloligonucleosomes. In a related embodiment, spacer arms are introducedbetween the solid support and 5′ adapter to improve ligation of thetarget nucleic acid to the adapter molecule. In another relatedembodiment, the ligated sample bound to a solid support is combined witha ligated sample that does not have a solid support prior to theamplification step.

The term “spacer arms” as used herein refers to any molecule that can beused in single or multiples to create space between the solid supportand an oligonucleotide (e.g., target nucleic acid). In a preferredembodiment of the invention, one or more hexaethylene (HEG) spacer unitsare inserted between the aminohexyl groups and the 5′ end of the 5′adapter. The aminohexyl group is used for covalent coupling to the solidsupport. Other examples of spacer arms that may be used in the presentinvention include multiple dTTP's (up to 15), spacer 18 (an 18 atomhexa-ethylene glycol spacer), spacer 9 (a triethylene glycol spacer), orphotocleavable spacers known in the art.

An alternative method of the present invention selectively detects andamplifies target nucleic acid using a combination of the following 3steps:

1) treating total isolated DNA from a biological sample with a ligasethat can covalently join blunt 5′-phosphorylated DNA ends (e.g. T4 or T7DNA ligase) under conditions that favor unimolecular circularization ofthe DNA molecules;

2) amplifying the DNA with target-specific primers and a method that isselective for circular DNA, for example, either a) via a rolling circleamplification with target-specific primers, or b) via inverse PCR withtarget-specific primers for the gene of interest; and3) characterizing the amplified DNA by direct or indirect qualitativeand/or quantitative molecular characterization methods, such as: a)Sequenom Inc.'s primer extension method (e.g., iPLEX™), orb) any knownmethod for detection and quantitation of nucleic acids such as DNAsequencing, restriction fragment length polymorphism (RFLP analysis),allele specific oligonucleotide (ASO) analysis, methylation-specific PCR(MSPCR), pyrosequencing analysis, acycloprime analysis, Reverse dotblot, GeneChip microarrays, Dynamic allele-specific hybridization(DASH), Peptide nucleic acid (PNA) and locked nucleic acids (LNA)probes, TaqMan, Molecular Beacons, Intercalating dye, FRET primers,AlphaScreen, SNPstream, genetic bit analysis (GBA), Multiplexminisequencing, SNaPshot, GOOD assay, Microarray miniseq, arrayed primerextension (APEX), Microarray primer extension, Tag arrays, Codedmicrospheres, Template-directed incorporation (TDI), fluorescencepolarization, Colorimetric oligonucleotide ligation assay (OLA),Sequence-coded OLA, Microarray ligation, Ligase chain reaction, Padlockprobes, and Invader assay, or combinations thereof.

Any combination of these 3 steps will selectively enrich thedouble-stranded, blunt-ended 5′-phosphorylated DNA such as DNA fromapoptotic ladders by several orders of magnitude over the DNA fragmentspresent in the biological sample that cannot by circularized due to lackof blunt ends and/or missing 5′-terminal phosphate groups and allow acomparison of its sequence with the wild-type sequence of the sameorganism or the host organism in case of a transplant or a maternalsequence in case of a pregnancy, for instance.

In a variation of the method, before step 2, an aliquot of the total DNAis either treated with methylation-sensitive or methylation-resistantenzymes or with chemicals that convert methylated bases into differentbases so that methylated bases in the apoptotic DNA fragments can becharacterized after step 2 and 3.

Diagnostic Applications

Circulating nucleic acids in the plasma and serum of patients areassociated with certain diseases and conditions (See, Lo Y M D et al., NEng J Med 1998; 339:1734-8; Chen X Q, et al., Nat Med 1996; 2:1033-5,Nawroz H et al., Nat Med 1996; 2:1035-7; Lo Y M D et al., Lancet 1998;351:1329-30; Lo Y M D, et al., Clin Chem 2000; 46:319-23). Further, themethod of nucleic acid isolation may affect the ability to detect thesedisease-associated nucleic acids circulating in the blood (Wang et al.Clin Chem. 2004 January; 50(1):211-3).

The characteristics and biological origin of circulating nucleic acidsare not completely understood. However, it is likely that cell death,including apoptosis, is one major factor (Fournie e al., Gerontology1993; 39:215-21; Fournie et al., Cancer Lett 1995; 91:221-7). Withoutbeing bound by theory, as cells undergoing apoptosis dispose nucleicacids into apoptotic bodies, it is possible that at least part of thecirculating nucleic acids in the plasma or serum of human subjects isshort, fragmented DNA that takes the form particle-associatednucleosomes. The present invention provides methods for extracting theshort, fragmented circulating nucleic acids present in the plasma orserum of subjects, thereby enriching the short, predictive nucleic acidsrelative to the background genomic DNA.

The present invention provides methods of evaluating a disease conditionin a patient suspected of suffering or known to suffer from the diseasecondition. In one embodiment of the present invention, the inventionincludes obtaining a biological sample from the patient suspected ofsuffering or known to suffer from a disease condition, selectivelyextracting and enriching extracellular nucleic acid in the sample basedon its size using the methods provided herein, and evaluating thedisease condition by determining the amount or concentration orcharacteristic of enriched extracellular nucleic acid and comparing theamount or concentration or characteristic of enriched extracellularnucleic acid to a control (e.g., background genomic DNA from biologicalsample).

The phrase “evaluating a disease condition” refers to assessing thedisease condition of a patient. For example, evaluating the condition ofa patient can include detecting the presence or absence of the diseasein the patient. Once the presence of disease in the patient is detected,evaluating the disease condition of the patient may include determiningthe severity of disease in the patient. It may further include usingthat determination to make a disease prognosis, e.g. a prognosis ortreatment plan. Evaluating the condition of a patient may also includedetermining if a patient has a disease or has suffered from a diseasecondition in the past. Evaluating the disease condition in that instantmight also include determining the probability of reoccurrence of thedisease condition or monitoring the reoccurrence in a patient.Evaluating the disease condition might also include monitoring a patientfor signs of disease. Evaluating a disease condition therefore includesdetecting, diagnosing, or monitoring a disease condition in a patient aswell as determining a patient prognosis or treatment plan. The method ofevaluating a disease condition aids in risk stratification.

Cancer

The methods provided herein may be used to extract oncogenic nucleicacid, which may be further used for the detection, diagnosis orprognosis of a cancer-related disorder. In plasma from cancer patients,nucleic acids, including DNA and RNA, are known to be present (Lo K W,et al. Clin Chem (1999) 45, 1292-1294). These molecules are likelypackaged in apoptotic bodies and, hence, rendered more stable comparedto ‘free RNA’ (Anker P and Stroun M, Clin Chem (2002) 48, 1210-1211; NgE K, et al. Proc Natl Acad Sci USA (2003) 100, 4748-4753).

In the late 1980s and 1990s several groups demonstrated that plasma DNAderived from cancer patients displayed tumor-specific characteristics,including decreased strand stability, Ras and p53 mutations,microsatellite alterations, abnormal promoter hypermethylation ofselected genes, mitochondrial DNA mutations and tumor-related viral DNA(Stroun M, et al. Oncology (1989) 46, 318-322; Chen X Q, et al. Nat Med(1996) 2, 1033-1035; Anker P, et al. Cancer Metastasis Rev (1999) 18,65-73; Chan K C and Lo Y M, Histol Histopathol (2002) 17, 937-943).Tumor-specific DNA for a wide range of malignancies has been found:haematological, colorectal, pancreatic, skin, head-and-neck, lung,breast, kidney, ovarian, nasopharyngeal, liver, bladder, gastric,prostate and cervix. In aggregate, the above data show thattumor-derived DNA in plasma is ubiquitous in affected patients, andlikely the result of a common biological process such as apoptosis.Investigations into the size of these plasma DNA fragments from cancerpatients has revealed that the majority show lengths in multiples ofnucleosomal DNA, a characteristic of apoptotic DNA fragmentation (JahrS, et al. Cancer Res (2001) 61, 1659-1665).

If a cancer shows specific viral DNA sequences or tumor suppressorand/or oncogene mutant sequences, PCR-specific strategies can bedeveloped. However, for most cancers (and most Mendelian disorders),clinical application awaits optimization of methods to isolate, quantifyand characterize the tumor-specific DNA compared to the patient's normalDNA, which is also present in plasma. Therefore, understanding themolecular structure and dynamics of DNA in plasma of normal individualsis necessary to achieve further advancement in this field.

Thus, the present invention relates to detection of specificextracellular nucleic acid in plasma or serum fractions of human oranimal blood associated with neoplastic, pre-malignant or proliferativedisease. Specifically, the invention relates to detection of nucleicacid derived from mutant oncogenes or other tumor-associated DNA, and tothose methods of detecting and monitoring extracellular mutant oncogenesor tumor-associated DNA found in the plasma or serum fraction of bloodby using DNA extraction with enrichment for mutant DNA as providedherein. In particular, the invention relates to the detection,identification, or monitoring of the existence, progression or clinicalstatus of benign, premalignant, or malignant neoplasms in humans orother animals that contain a mutation that is associated with theneoplasm through the size selective enrichment methods provided herein,and subsequent detection of the mutated nucleic acid of the neoplasm inthe enriched DNA.

The present invention features methods for identifying DNA originatingfrom a tumor in a biological sample. These methods may be used todifferentiate or detect tumor-derived DNA in the form of apoptoticbodies or nucleosomes in a biological sample. In preferred embodiments,the non-cancerous DNA and tumor-derived DNA are differentiated byobserving nucleic acid size differences, wherein low base pair DNA isassociated with cancer.

Prenatal Diagnostics

Since 1997, it is known that free fetal DNA can be detected in the bloodcirculation of pregnant women. In absence of pregnancy-associatedcomplications, the total concentration of circulating DNA is in therange of 10-100 ng or 1,000 to 10,000 genome equivalents/ml plasma(Bischoff et al., Hum Reprod Update. 2005 January-February; 11(1):59-67and references cited therein) while the concentrations of the fetal DNAfraction increases from ca. 20 copies/ml in the first trimester to >250copies/ml in the third trimester. After electron microscopicinvestigation and ultrafiltration enrichment experiments, the authorsconclude that apoptotic bodies carrying fragmented nucleosomal DNA ofplacental origin are the source of fetal DNA in maternal plasma.

It has been demonstrated that the circulating DNA molecules aresignificantly larger in size in pregnant women than in non-pregnantwomen with median percentages of total plasma DNA of >201 bp at 57% and14% for pregnant and non-pregnant women, respectively while the medianpercentages of fetal-derived DNA with sizes >193 bp and >313 bp wereonly 20% and 0%, respectively (Chan et al, Clin Chem. 2004 January;50(1):88-92).

These findings have been independently confirmed (Li et al, Clin Chem.2004 June; 50(6):1002-11); Patent application US200516424, which ishereby incorporated by reference) who showed as a proof of concept, thata >5 fold relative enrichment of fetal DNA from ca. 5% to >28% of totalcirculating plasma DNA is possible be means of size exclusionchromatography via preparative agarose gel electrophoresis and elutionof the <300 bp size fraction. Unfortunately, the method is not verypractical for reliable routine use because it is difficult to automateand due to possible loss of DNA material and the low concentration ofthe DNA recovered from the relevant Agarose gel section.

Thus, the present invention features methods for differentiating DNAspecies originating from different individuals in a biological sample.These methods may be used to differentiate or detect fetal DNA in amaternal sample. In preferred embodiments, the DNA species aredifferentiated by observing nucleic acid size differences.

The differentiation between maternal and fetal DNA may be performed withor without quantifying the concentration of fetal DNA in maternal plasmaor serum. In embodiments wherein the fetal DNA is quantified, themeasured concentration may be used to predict, monitor or diagnose orprognosticate a pregnancy-associated disorder.

There are a variety of non-invasive and invasive techniques availablefor prenatal diagnosis including ultrasonography, amniocentesis,chorionic villi sampling (CVS), fetal blood cells in maternal blood,maternal serum alpha-fetoprotein, maternal serum beta-HCG, and maternalserum estriol. However, the techniques that are non-invasive are lessspecific, and the techniques with high specificity and high sensitivityare highly invasive. Furthermore, most techniques can be applied onlyduring specific time periods during pregnancy for greatest utility

The first marker that was developed for fetal DNA detection in maternalplasma was the Y chromosome, which is present in male fetuses (Lo et al.Am J Hum Genet (1998) 62:768-775). The robustness of Y chromosomalmarkers has been reproduced by many workers in the field (Costa J M, etal. Prenat Diagn 21:1070-1074). This approach constitutes a highlyaccurate method for the determination of fetal gender, which is usefulfor the prenatal investigation of sex-linked diseases (Costa J M,Ernault P (2002) Clin Chem 48:679-680).

Maternal plasma DNA analysis is also useful for the noninvasive prenataldetermination of fetal RhD blood group status in RhD-negative pregnantwomen (Lo et al. (1998) N Engl J Med 339:1734-1738). This approach hasbeen shown by many groups to be accurate, and has been introduced as aroutine service by the British National Blood Service since 2001(Finning K M, et al. (2002) Transfusion 42:1079-1085).

More recently, maternal plasma DNA analysis has been shown to be usefulfor the noninvasive prenatal exclusion of fetal β-thalassemia major(Chiu R W K, et al. (2002) Lancet 360:998-1000). A similar approach hasalso been used for prenatal detection of the HbE gene (Fucharoen G, etal. (2003) Prenat Diagn 23:393-396).

Other genetic applications of fetal DNA in maternal plasma include thedetection of achondroplasia (Saito H, et al. (2000) Lancet 356:1170),myotonic dystrophy (Amicucci P, et al. (2000) Clin Chem 46:301-302),cystic fibrosis (Gonzalez-Gonzalez M C, et al. (2002) Prenat Diagn22:946-948), Huntington disease (Gonzalez-Gonzalez M C, et al. (2003)Prenat Diagn 23:232-234), and congenital adrenal hyperplasia (Rijnders RJ, et al. (2001) Obstet Gynecol 98:374-378). It is expected that thespectrum of such applications will increase over the next few years.

In another aspect of the present invention, the patient is pregnant andthe method of evaluating a disease or physiological condition in thepatient or her fetus aids in the detection, monitoring, prognosis ortreatment of the patient or her fetus. More specifically, the presentinvention features methods of detecting abnormalities in a fetus bydetecting fetal DNA in a biological sample obtained from a mother. Themethods according to the present invention provide for detecting fetalDNA in a maternal sample by differentiating the fetal DNA from thematernal DNA based on DNA characteristics (e.g., size, weight, 5′phosphorylated, blunt end). See Chan et al. Clin Chem. 2004 January;50(1):88-92; and Li et al. Clin Chem. 2004 June; 50(6):1002-11.Employing such methods, fetal DNA that is predictive of a geneticanomaly or genetic-based disease may be identified thereby providingmethods for prenatal diagnosis. These methods are applicable to any andall pregnancy-associated conditions for which nucleic acid changes,mutations or other characteristics (e.g., methylation state) areassociated with a disease state. Exemplary diseases that may bediagnosed include, for example, preeclampsia, preterm labor, hyperemesisgravidarum, ectopic pregnancy, fetal chromosomal aneuploidy (such astrisomy 18, 21, or 13), and intrauterine growth retardation.

The compositions, methods and kits of the present invention allow forthe analysis of fetal genetic traits including those involved inchromosomal aberrations (e.g. aneuploidies or chromosomal aberrationsassociated with Down's syndrome) or hereditary Mendelian geneticdisorders and, respectively, genetic markers associated therewith (e.g.single gene disorders such as cystic fibrosis or thehemoglobinopathies). Size-based extraction of extracellular fetal DNA inthe maternal circulation thus facilitates the non-invasive detection offetal genetic traits, including paternally inherited polymorphisms whichpermit paternity testing.

The term “pregnancy-associated disorder,” as used in this application,refers to any condition or disease that may affect a pregnant woman, thefetus the woman is carrying, or both the woman and the fetus. Such acondition or disease may manifest its symptoms during a limited timeperiod, e.g., during pregnancy or delivery, or may last the entire lifespan of the fetus following its birth. Some examples of apregnancy-associated disorder include ectopic pregnancy, preeclampsia,preterm labor, and fetal chromosomal abnormalities such as trisomy 13,18, or 21.

The term “chromosomal abnormality” refers to a deviation between thestructure of the subject chromosome and a normal homologous chromosome.The term “normal” refers to the predominate karyotype or banding patternfound in healthy individuals of a particular species. A chromosomalabnormality can be numerical or structural, and includes but is notlimited to aneuploidy, polyploidy, inversion, a trisomy, a monosomy,duplication, deletion, deletion of a part of a chromosome, addition,addition of a part of chromosome, insertion, a fragment of a chromosome,a region of a chromosome, chromosomal rearrangement, and translocation.A chromosomal abnormality can be correlated with presence of apathological condition or with a predisposition to develop apathological condition.

Other Diseases

Many diseases, disorders and conditions (e.g., tissue or organrejection) produce apoptotic or nucleosomal DNA that may be detected bythe methods provided herein. Diseases and disorders believed to produceapoptotic DNA include diabetes, heart disease, stroke, trauma andrheumatoid arthritis. Lupus erythematosus (SLE) (Rumore and Steinman JClin Invest. 1990 July; 86(1):69-74). Rumore et al. noted that DNApurified from SLE plasma formed discrete bands, corresponding to sizesof about 150-200, 400, 600, and 800 bp, closely resembling thecharacteristic 200 bp “ladder” found with oligonucleosomal DNA.

The present invention also provides a method of evaluating the diseasecondition of a patient suspected of having suffered from a trauma orknown to have suffered from a trauma. The method includes obtaining asample of plasma or serum from the patient suspected of having sufferedfrom a trauma or known to have had suffered from a trauma, and detectingthe quantity or concentration of mitochondrial nucleic acid in thesample.

EXAMPLES

The examples hereafter illustrate but do not limit the invention.

Example 1 Isolation of DNA Using a Double Extraction Salt-Based Method

The example provides a procedure, using a method provided herein, toselectively extract DNA based on its size.

1. Protein Denaturation and Protein Digestion

Add a low concentration of chaotropic salt, for example, less than 30%solution (weight per volume) to the sample solution to denature proteinsand inactivate nucleases, proteinase K or any protease, for example 100to 1000 μg) to further inactivate nucleases and break down proteins insolution. Alternatively, detergents, for example SDS or Triton-X 100 upto 1% volume per volume, may be used alone or in combination with asalt.

Mix the solution thoroughly, and incubate for 10-30 minutes at 55° C.,or sufficient time and temperature for the enzyme in use.

2. Binding of Non-Target Nucleic Acid

Add a low concentration of salt, for example, 10-30% weight per volume,and add the solid support.

Mix the solution thoroughly, and incubate 10-30 minutes at ambienttemperature.

3. Separate of the Solid Support from the Solution

Transfer the solid support or solution from the vessel to a new vessel.

4. Binding of Target Nucleic Acid

Add a high concentration of salt, for example, 20-60% weight per volume,and add (fresh) solid support.

Add a Carrier Complex.

Mix the solution thoroughly, and incubate 10-30 minutes at ambienttemperature.

5. Separate the Solid Support from the Solution

Discard the supernatant and proceed to washing the target nucleicacid-bound solid support.

6. Washing

Wash target nucleic acid-bound solid support using an appropriatewashing solution comprised of salt, buffer, water and alcohol. (Additionof carrier to wash solution may increase recovery).

Remove washing solution and repeat by gradually increasing alcoholconcentration in the wash solution.

7. Air Dry

Air dry solid support at ambient temperature or by exposing to heat tocompletely dry and remove any remaining alcohol that would inhibitdownstream use of the sample.

8. Elution

Release the target nucleic acid from the solid support by addition ofsufficient sterile water or buffered solution (e.g. 1×TE pH 7-8.5) atambient temperature or by exposing to heat.

9. Collect Elute Containing Targeted Nucleic Acid.

Example 2 DNA Extraction in the Presence of Guanidine Thiocyanate(GuSCN)

FIG. 1 shows the successful extraction of low base pair DNA from a 1 kbDNA ladder (Promega™) in the presence of guanidine thiocyanate (GuSCN).The DNA is first bound to silica at various low concentrations ofguanidine thiocyanate as shown in FIG. 1. The supernatant from the firstbinding solution is subsequently bound to silica at varying guanidinethiocyanate concentrations, with a finishing high concentration of 4.6M.These steps are followed by wash and elution steps.

The method can be employed to produce size selective separation of acommercially available DNA ladder with DNA strands ranging in mass from250 bp to 10,000 bp from normal human plasma in the presence ofguanidine thiocyanate as the chaotropic salt. The following steps areperformed:

1. To four separate vessels, add 10 μL protease solution (20 ug/μL), 200μL normal human plasma, and 100 μL 4.5 M GuSCN (final concentration 1.45M).

2. Add GuSCN sufficient to increase the concentration to 4M, 3M, 2.5M or2M. Add 5 μL hydrated silica, 10 μL 1 kb ladder. Mix the solution andincubated 10 minutes at ambient temperature.

3. Centrifuge at 7,000 rpm for 2.5 minutes. Transfer the supernatant toa new vessel. Label the non-target DNA silica as E1 and proceed towashing step (Step 6).

4. Increase the concentration of the supernatant to 4.6 M GuSCN for allsamples. Add 5 μL of silica, mix and incubate for 10 minutes at ambienttemperature.

5. Centrifuge at 7,000 rpm for 2.5 minutes. Discard the supernatant.Label the target DNA silica as E2 and proceed to washing step (Step 6).

6. Wash pellet 1×500 μL of 2 M GuSCN in 67% ethanol, 1×500 μL of 1MGuSCN in 83% ethanol, 1×100 μl ethanol.

7. Air dry silica 5 minutes on low heat.

8. Add 10 μL of 1×TE and incubate at 55 degrees C. for 10 minutes.

9. Transfer eluates for E1 and E2 to new tube. Add 2.5 μL bromophenolblue loading buffer, and load 10 μL per lane onto a 1.2% agarose gel in1×TBE. Electrophoresis is used to separate the DNA strands, and ethidiumbromide is used to visualize the DNA. FIG. 1 shows results of themethod.

Example 3 DNA Extraction in the Presence of Sodium Perchlorate (NaClO₄)

FIG. 2 shows the successful extraction of low base pair DNA from a 1 kbDNA ladder (Promega™) in the presence of sodium perchlorate (NaClO₄).The DNA is first bound to silica at various low concentrations of sodiumperchlorate as shown in FIG. 2. The supernatant from the first bindingsolution is subsequently bound to silica at varying sodium perchlorateconcentrations, with a finishing high concentration of 4.5 M. Thesesteps are followed by wash and elution steps.

The method can be employed to produce size selective separation of acommercially available DNA ladder with DNA strands ranging in mass from250 bp to 10,000 bp from normal human plasma in the presence of sodiumperchlorate (NaClO₄) as the chaotropic salt. The following steps areperformed:

1. To four separate vessels, add 10 μL protease solution (20 ug/ul), 200μL normal human plasma, and 100 μL 4.5 M GuSCN (final concentration 1.45M).

2. Add NaClO₄ to final concentrations of 3M, 2M, 1.5M or 1M. Add 5 μLhydrated silica, 10 μL 1 kb ladder. Mix the solution and incubate 10minutes at ambient temperature.

3. Centrifuge at 7,000 rpm for 2.5 minutes. Transfer the supernatant toa new vessel. Label the non-target DNA silica as E1 and proceeded towashing step (Step 6).

4. Increase the concentration of the supernatant to 4.5 M NaClO₄ for allsamples. Add 5 μL of silica, mix and incubate for 10 minutes at ambienttemperature.

5. Centrifuge at 7,000 rpm for 2.5 minutes. Discard the supernatant.Label the target DNA silica as E2 and proceeded to washing step (Step6).

6. Wash pellet 2×500 μL of 1.2 M NaClO₄ in 70% ethanol, 1×100 μlethanol.

7. Air dry silica 5 minutes on low heat.

8. Add 10 μL of 1×TE and incubate at 55 C for 10 minutes.

9. Transfer eluates for E1 and E2 to new tube. Add 2.5 μL bromophenolblue loading buffer, and loaded 10 μL per lane onto a 1.2% agarose gelin 1×TBE. Electrophoresis is used to separate the DNA strands, andethidium bromide is used to visualize the DNA. FIG. 2 shows results ofthe method.

Example 4 Adapter-Mediated Ligation of Target Nucleic Acid

The below example provides a procedure, using the method providedherein, to selectively amplify target nucleic acid that is blunt-endedand 5′-phosphorylated. The method relies on the ligation of a non-genomespecific adapter to the blunt ends, which allows for whole genomeamplification followed by target-specific amplification. While thenature of the termini of all cell-free nucleic acid is unknown, couplingthis method with short extension times during amplification will favorthe amplification of the oligonucleosome monomer and short multimers.Since the target nucleic acid is shorter than the non-targeted nucleicacid, the target nucleic acid can be enriched over the non-targetnucleic acid. This method can be further coupled with specificamplification of a nucleic acid region of interest for further analysis.FIG. 3 shows results of the method.

Exemplary Adapters:

(SEQ ID NO: 1) 5′ adapter: 5′-ACACGGCGCACGCCTCCACG-3′ (SEQ ID NO: 2) 3′adapter: 5′-CGTGGAGGCGTG-3′The 3′ adapter is complementary to the 3′-end of 5′ adapter to createthe blunt-end, double-stranded adapter complex.

(SEQ ID NO: 1) 5′ adapter: 5′-ACACGGCGCACGCCTCCACG-3′ } blunt-end(SEQ ID NO: 2) 3′ adapter: 3′-GTGCGGAGGTGC-5′Alternatively, the 3′ adapter molecule is modified such that the newsequence consists of 13 nucleobases with a dideoxy-nucleotide at its3′-position. This terminator nucleotide does not allow extension of the3′ adapter molecule by any polymerase, thus improving assay efficiencyand detection.Alternative 3′ Adapter:

(SEQ ID NO: 3) 3′ adapter (SSGA13dd3′): 5′-CGTGGAGGCGTGddNTP-3′The 3′ adapter is shorter to reduce the melting temperature and allowfor release from the 5′ adapter following ligation. Both adapters arenon-phosphorylated, and may be made of any sequence that is nonspecificto the nucleic acid to be amplified to prevent non-specificamplification of the genome.An exemplary procedure is provided below:

1. Prepare total or size selective nucleic acid sample in water orbuffer.

2. Add ligation buffer, 5′ adapter, 3′ adapter, and water to reactionvolume.

3. Place the reaction into a thermocycler and heat the reaction to 55°C. for 10 minutes, then slowly ramp down the temperature to 10° C. over1 hour.

4. Add 1 μl T4 ligase (1-3 U per ul) (or ligation enzyme) and mix welland incubate for 10 min at 10° C., then ramp temperature up to 16° C.and incubate for 10 minutes to over night (12-16 hours).

5. Add 5′ primer, 10×PCR buffer, MgCl₂, dNTPs, and polymerase.

6. Incubate at 72° C. for 10 minutes (displacement of 3′ adapter andinitial extension of the ligated sample)

7. Thermocycle for non-template specific amplification

Template Denaturation 10 sec at 94° C. 5′ adapter primer annealing 10sec at 56° C. 5′ adapter primer Extension 10 sec at 72° C. Finalextension 1 min at 72° C. Hold at 4° C.

8. Add template-specific 5′ and 3′ primers, 10×PCR buffer, MgCl₂, dNTPs,and polymerase.

9. Continue with sample to perform target-specific amplification anddetection.

Example 5 Rolling-Circle Amplification of Target Nucleic Acid

Described hereafter is a method for intramolecular ligation followed byamplification of a target by inverse PCR or rolling circle amplificationto detect a target nucleic acid.

Inverse Amplification (See FIG. 4)

1. Prepare total or size selective nucleic acid sample in water orbuffer.

2. Add ligation buffer, 1 μL ligase (1-10 U per μL) and water toreaction volume.

3. Mix well and incubate for 10 minutes to over night (12-16 hours) at4-25° C., followed by ligase inactivation at 65 C for 10 min (or asrequired for enzyme used).

4. Add PCR buffer, dNTPs, MgCl, inverse PCR primers, and polymerase tothe sample.

5. Thermocycle for 45 cycles non-template specific amplification

Template Denaturation 20 sec at 94° C. 5′ primer Annealing 15 sec at 56°C. 5′ primer Extension 10 sec at 72° C. Hold at 4° C.Rolling Circle Amplification (See FIG. 5)1. Prepare total or size selective nucleic acid sample in water orbuffer.2. Add ligation buffer, 1 μL ligase (1-10 U per μL) and water toreaction volume.3. Mix well and incubate for 10 minutes to over night (12-16 hours) at4-25° C., followed by ligase inactivation at 65° C. for 10 min (or asrequired for enzyme used).4. Add target specific forward and reverse primers, heat the reaction to95° C. for 3 minutes to denature the template, then rapidly cool to 4°C. (on ice).5. Add reaction buffer, dNTP, and strand displacing polymerase (e.g.Templi Phi (Amersham).6. Elongate at 30° C. for 12 hours or more7. Stop the reaction by heating to 65° C. for 10 minutes8. Proceed to detection method

The entirety of each patent, patent application, publication anddocument referenced herein hereby is incorporated by reference. Citationof the above patents, patent applications, publications and documents isnot an admission that any of the foregoing is pertinent prior art, nordoes it constitute any admission as to the contents or date of thesepublications or documents.

Modifications may be made to the foregoing without departing from thebasic aspects of the invention. Although the invention has beendescribed in substantial detail with reference to one or more specificembodiments, those of ordinary skill in the art will recognize thatchanges may be made to the embodiments specifically disclosed in thisapplication, and yet these modifications and improvements are within thescope and spirit of the invention. The invention illustrativelydescribed herein suitably may be practiced in the absence of anyelement(s) not specifically disclosed herein. Thus, for example, in eachinstance herein any of the terms “comprising”, “consisting essentiallyof”, and “consisting of” may be replaced with either of the other twoterms. Thus, the terms and expressions which have been employed are usedas terms of description and not of limitation, equivalents of thefeatures shown and described, or portions thereof, are not excluded, andit is recognized that various modifications are possible within thescope of the invention. Embodiments of the invention are set forth inthe following claims.

What is claimed is:
 1. A method for extracting low molecular weightapoptotic DNA from a cell-free DNA sample comprising a mixture of lowmolecular weight DNA and high molecular weight DNA, which comprises: (a)mixing the sample, guanidine (iso)thiocyanate or sodium perchlorate, anda silica solid support comprising silica capable of reversibly bindingDNA, thereby forming a first binding solution, wherein the guanidine(iso)thiocyanate or sodium perchlorate is present at 1-4M, at which thehigh molecular weight DNA selectively adsorbs to the solid support; (b)separating the silica solid support from the first binding solution,thereby yielding a fraction separated from the solid support; (c) mixingthe fraction separated from the silica solid support in (b) withadditional guanidine (iso)thiocyanate or sodium perchlorate, whereby theguanidine (iso)thiocyanate or sodium perchlorate is present at a higherconcentration than that of the first binding solution in (a); (d)introducing additional silica solid support to the mixture in (c),thereby forming a second binding solution, wherein the low molecularweight DNA selectively adsorbs to the additional solid support; and (e)separating the additional silica solid support from the second bindingsolution, thereby extracting low molecular weight DNA from the sample,wherein the low molecular weight DNA comprises less than 1200 base pairsand the high molecular weight DNA comprises 1200 or more base pairs. 2.The method of claim 1, wherein the sample is blood serum, blood plasmaor urine.
 3. The method of claim 1, further comprising eluting theadsorbed DNA from the separated solid support in (e).
 4. The method ofclaim 2, wherein the blood serum, blood plasma or urine is from apregnant human.
 5. The method of claim 1, wherein the low molecularweight DNA is of fetal origin.
 6. The method of claim 1, wherein thehigh molecular weight DNA is of maternal origin.
 7. The method of claim1, wherein the silica solid support comprises a silica coated magneticbead.
 8. The method of claim 1, wherein the guanidine (iso)thiocyanateor sodium perchlorate of (a) is present at a concentration in the rangeof 10% to 30% weight and the guanidine (iso)thiocyanate or sodiumperchlorate of (c) is present at a concentration in the range of 20% to60% weight.